The Development And Application Of Brian Organoids And Mechanism Studies Of Matrine In The Treatment Of Glioblastoma

Background:Gliomas originated from glial cells are a heterogeneous group of neoplasia that account for the majority of primary intracranial tumors.Glioblastoma multiforme(GBM)represents the most common and biologically aggressive malignant subtype of glioma.Invasion is one of primary characteristics of GBM.Extensive tumor cells that invade into the brain parenchyma is one of the main reasons leading to high recurrence and poor prognosis of GBM,which is difficult to be completely removed during surgery and subsequent radiotherapy/temozolomide-based chemotherapy.Therefore,to establish a good experimental research model,and then use this model to isolate invasive glioma cells,and further to develop new drugs targeting invasive cells has substantial clinical transformation value.In vitro tumor cell culture model is mainly used by the laboratory to study the invasion of glioma cells and its mechanism,including wound healing assay,cell invasion into various extracellular matrix gels(transwell assay,3D tumor spheroids invasion assay,etc.)and cell migration on brain slices.However,the invasion and migration of tumor is a very complex process involving multiple internal and external factors,and the changes caused by tumor cells and tumor microenvironment(TME)are the key processes.TME comprises tumor stromal,adjacent cells,vessel,surrounding immune cells and molecules.Changes of cellular components in the tumor microenvironment play a vital role in inter-and intra-tumor cell heterogeneity,as well as cancer malignant proliferation,invasion,and angiogenesis of tumors.Cancer-associated fibroblasts(CAFs)can induce epithelial mesenchymal transition(EMT)and tumor stem cell phenotype of breast cancer cells,and significantly promote the invasion and metastasis of breast cancer.Our study also showed that CAFs can directly affect the metabolic process of colorectal cancer through the anti-Warburg effect,thereby promoting the malignant proliferation,invasion and metastasis of tumors.Moreover,studies in the glioma microenvironment have showed that reactive glioma-associated astrocytes markedly upregulate glioma cell invasion by highly expressing connexin 43 Glioma-associated microglial cells(GAMs)are closely related to the formation of glioma immunosuppressive microenvironment,leading to drug resistance in GBM.Therefore,it is highly recognized that the tumor microenvironment plays a crucial role in the occurrence,progression,invasion and the reactivity to GBM.However,the traditional in vitro models used do not contain various components of the tumor microenvironment,which are difficult to reflect the complex structural and functional characteristics of the brain environment in vivo.Through the IVYGAP project(http://glioblastoma.alleninstitute.org),RNA-Seq data are currently available for>40 GBMs where tissue samples have been collected from different tumor regions including the leading edge and infiltrative tumor sites.However,it is difficult to obtain an exact signature since it is frequently difficult by histopathology,to decipher invasive tumor cells from reactive glial cells.Therefore,it is needed to establish a reliable ex-vivo system to mimic brain tumor invasion,where the tumor cells can invade into a mature differentiated brain structure,and their morphological,future genomics and proteomics assessment of tumor cell invasion can be studied in real time.In this context,we firstly developed a brain organoids model that can mimic the brain tumor microenvironment and explained in detail the process of its development and maturation into mature brain organoids.Secondly,we co-cultured our mature brain organoids with patient-derived primary GBM spheroids models and tracked the movement of GFP labeled GBM cells in brain organoids of the co-culture system in real time by confocal microscope,and determined the invasion capacity and invasion speed of tumor cells.Thirdly,we tested a novel treatment strategy matrine that may be target GBM using co-culture system,and explored the therapeutic effect and mechanism of matrine.PART ?.Establishment of a novel ex-vivo brain organoids modelObjectTo develop a novel ex-vivo brain organoids model and study in detail the process of differentiation and development of various neural cell lineages into mature brain structures.Methods1.These organoids are made by the removal of brain tissue from rat fetuses at 18 days of gestation,whereupon the cells are dissociated and reaggregated into fetal brain organoid structures in vitro.2.Western blotting(WB)was used to detect neural stem cell markers(Nestin,SOX2)and mature neuron markers(NeuN),astrocyte markers(GFAP),microglial markers(CD11b,AIF-1)and oligodendrocyte markers(MBP,Oligo2)in brain organoids at different developmental stages(Day 4,10,14 and 21).3.Hematoxylin and eosin(H&E)staining was used to detect structural organization of brain organoids at different developmental stages and immunohistochemistry(IHC)were used to detect the expression and distribution of mature neurons,astrocytes,oligodendrocyte in the mature brain organoids.4.Transmission electron microscopy(TEM)was used to analyze the development status of neuron in 21-day brain organoids.5.RNA-seq and protein analysis were used to evaluate the development of brain organoids and compared with the development of induced pluripotent stem cells(IPSc)derived brain organoids and the postnatal rat brain obtained from the database.Results1.WB analysis revealed a consistent reduction of the stem cell markers(Nestin,SOX2)during brain organoids development,whereas,mature neuron markers(NeuN),astrocyte markers(GFAP),oligodendrocyte markers(MBP)and Microglia markers(CD11b,AIF-1)were significantly upregulated.21-day brain organoids have the highest expression levels.2.H&E staining results indicated that the 21-day brain organoids had obvious structural divisions:white matter area and gray matter area.3.IHC results show different cell localizations in 21-day brain organoids:astrocytes are mainly distributed in white matter and surface layers;neurons are mainly distributed in gray matter;microglia are scattered in gray matter and superficial area;oligodendrocytes are distributed in superficial area.The 21-day brain organoids showed a highly cellular and structural organization.4.TEM revealed numerous synapses as well as myelinated axons within 21-day brain organoids.5.Rat brain organoid development shows strong similarities to rat brain development in vivo at the transcriptomic level.A global comparison of expression data revealed a strong correlation between organoids at day 21 and rat brains postnatal day 21.These results indicate that the 21-day brain organoids are mature brain organoids.6.Proteomics reveal,during differentiation,both similar expression patterns as well as skewed patterns when compared to transcriptomics.7.Protein expression profiles analysis showed that four months old human IPSc brain organoids correspond to day-10 rat brain organoid development.This implies that rat brain organoid represents a better model for invasion studies compared to 4-months old IPSc Brain organoids,which at this stage still represent developmental brain structures where numerous developmental programs are up-regulated(more similar to rat brain development postnatal day 10).ConclusionsWe have successfully developed an ex-vivo brain organoids model that is highly reproducible and mimics the situation in the postnatally mature brain.We also assessed in detail the differentiation of this model into mature brain organoids structures,providing a better in vitro research model for brain development and brain tumor research.PART ?.Application of a novel brain organoids model in glioblastoma researchObjectEstablish a co-culture system of brain organoids models and patient-derived primary glioma spheroids models to study glioblastoma(GBM)invasion and test new therapeutic strategies that may target GBM using this system.Methods1.Five patient-derived GBM cell lines(BG5,BG7,P3,GG16,GG6)developed in our lab were labeled by GFP lentivirus.2.Tumor cells were cultured as tumor spheroids using round bottom low attachment 96 well plates and co-cultured with our mature brain organoids.3.Confocal microscope was used to observe the invasion of GFP-labeled GBM cells in real time.4.Imaris software was used to track the trajectory of each GBM invasion cell and then the invasion speed of this cell can be calculated.5.Five patient-derived glioma cell lines were implanted into the brain of nude rats.We monitored the weight of nude rats daily,anesthetized the nude rats when the weight dropped by more than 20%and took the brain out after 4%paraformaldehyde infusion.And the brain was made into paraffin-embedded sections.H&E Staining was used to detect the invasion of different tumor cell lines.6.TGF? inhibitor has been reported to increase anti-migratory/anti-invasive as well as anti-angiogenic effects in GBM,we next evaluated TGF? inhibitor as a therapeutic compound using this well-established ex-vivo invasion model treated with TGF? factor and TGF? inhibitor to the co-culture system.7.The co-culture system was used to evaluate the effects of some drugs(matrine,albendazole,temozolomide,and thioridazine)in our lab on the invasion capacity of GBM cellsResults1.Different GBM cell lines showed different invasion capabilities and invasion speeds.Among the 5 patient-derived glioma cell lines developed in our lab,BG5 has the highest invasion ability and the fastest invasion speed,P3 is the invasive cell line,and the BG7 and GG16 cell lines have moderate invasion ability and invasion speed,while GG6 Cell line invasion is the least one.2.H&E staining results showed that the invasion ability of GBM in vivo was similar to that obtained in co-culture system.These experiments demonstrated the heterogeneities of GBM tumor invasion patterns within the brain organoids and thus the co-culture system highly mimic in vivo model.3.TGF? significantly enhanced the invasion capacity and increased the speed of tumor cells on BG7 cells,whereas TGF? inhibitors induce a marked inhibition.These data indicated that in vitro co-culture system can be used to detect the effect of cytokines and potential therapeutic strategies on invasive GBM.4.Temozolomide and matrine can significantly inhibit the invasion ability of GBM cells in the co-culture system.Matrine may be a potential therapeutic strategy towards to GBM.ConclusionsWe established a co-culture system of brain organoids models and GBM spheroids model to explore the invasion capacities of different GBM cells,and verify the effect of TGF? and TGF? inhibitors on invasion of GBM.We confirmed that co-culture system offers us an ex-vivo model which highly mimics in vivo model that will be providing experimental studies on developing cytokines and potential therapeutic strategies toward to invasive GBM.And matrine may be a potential drug towards to GBM patients.PART ?.The effect and mechanism studies of matrine in the treatment of glioblastomaObjectTo further determine the therapeutic effects and potential mechanism of matrine in the treatment of glioblastoma(GBM)Methods1.Cell counting kit-8(CCK-8)was used to detect the toxic effects of matrine on normal glial cells and GBM cell lines and to further determine the safe concentration of the drug.2.The effects of matrine on the proliferation of GBM cells were investigated using EdU proliferation assay.3.Wound healing assay and transwell invasion assay were used to verify the inhibitory effect of matrine on the invasion of GBM cells.4.Apoptosis of GBM cells induced by matrine was detected by flow cytometry using PI and V-FITC staining.5.TUNNEL staining was also used to detect the apoptosis of GBM cells induced by matrine.6.The effect of matrine on the cell cycle of GBM was measured by PI staining and flow cytometry.7.?H2AX immunofluorescence staining was used to detect matrine-induced DNA damage in GBM cells.8.SA-?-gal senescence staining kit was used to detect matrine-induced GBM cell senescence.9.WB was used to investigate the apoptosis-related proteins(PARP,Cleaved PARP,Caspase 3,Cleaved Caspase-3)and cycle-related proteins(Rb,p53,p21,p27,CKD4,CDK6)and pathway-related protein expression after matrine treated GBM cells at different times.10.Protein cytokine array,enzyme linked immunosorbent assay(Elisa),bioinformatics analysis and siRNA transfection experiments were used to determine the mechanism of action of matrine on GBM cells.11.The luciferase labeled GBM cell lines(U251,P3)were implanted into the brain of nude mice by stereotaxic apparatus,and PBS,matrine(40 mg/kg),matrine(80 mg/kg)were injected intraperitoneally every other day from the third day.Small animal imager was used to monitor the growth of intracranial tumors every week.The body weight of nude mice was measured every day.The mice were anesthetized when the body weight loss is?20%,and the brain were harvested after 4%paraformaldehyde infusion and made into paraffin sections.?-H2AX and p27 staining were used to investigate the effect of matrine on the proliferation and senescence of GBM in vivo.Results1.Matrine significantly inhibited proliferation of GBM cell lines.1.1 Dose-response curves based on cell viability data collected from the CCK-8 assay indicated that treatment with increasing concentrations of matrine resulted in growth inhibition of all cells tested.IC50 values at 72 hours for GBM cell lines were?1.5 to 2A?less than in NHA indicating that tumor cells were more sensitive to the alkaloid than NHA.This suggests that tumor cells are more sensitive to matrine than normal glial cells1.2 EdU results revealed a statistically significant decrease in proliferation in the matrine treated GBM cells.2.Wound healing assay and transwell invasion assay showed that matrine can significantly inhibit the invasion of GBM cells.3.Matrine induced senescence of GBM cell,but not apoptosis.3.1 Based on propidium iodide and annexin V staining and TUNNEL staining,the percentage of apoptotic cells did not increase significantly for either cell line treated with matrine.Moreover,the levels of two key proteins involved in apoptosis remained unchanged as assessed on Western blot.3.2 Cell cycle results showed that matrine induced GO/G1 phase arrest in GBM.3.3 Matrine treatment led to increased ?H2AX positive nuclei indicative of DNA double-strand breaks.3.4 Matrine treatment led to increased accumulation of SA-?-gal-positive cells,suggested that matrine induced cellular senescence of GBM cells.4.WB and siRNA transfection experiments determined that matrine induced cellular senescence of GBM cells by upregulating p27.5.Bioinformatics analysis and WB results identified that matrine inhibited the PI3K/AKT/p27 signaling pathway.6.Protein cytokine array and Elisa analysis revealed a reduction in secreted IGF1 from matrine-induced senescent GBM cells.WB results indicated that blocking of IGF1 led to decreased expression of PI3K/AKT/p27 signaling pathway.SA-P-gal senescence staining showed that downregulation of IGF 1 could enhance the effect of matrine induced GBM cells senescence.7.The in vivo data have confirmed that matrine c inhibit GBM by inducing senescence of tumor cells.7.1 Small animal imaging results showed that the intracranial tumor volume of nude mice treated with matrine was significantly reduced compared with the control group.7.2 Kaplan-Meier survival curve analysis showed that matrine could prolong the survival of GBM nude mice.7.3 IHC staining results showed that Ki67 positive cells in the tumor tissue of the matrine treated group significantly decreased,whereas yH2AX and p27 positive cells markedly increased compared with the control group.ConclusionsMatrine induced senescence of human GBM cells by through suppression of the IGF1/PI3K/AKT/p27 signaling pathway and further inhibited the proliferation and invasion of GBM.